Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides

Jan H. Kessler, Bregje Mommaas, Tuna Mutis, Ivo Huijbers, Debby Vissers, Willemien E. Benckhuijsen, Geziena M.Th Schreuder, Rienk Offringa, Els Goulmy, Cornelis J.M. Melief, Sjoerd H. Van der Burg, Jan W. Drijfhout

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We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membranebound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian pop ulation. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptidebinding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.

Original languageEnglish
Pages (from-to)245-255
Number of pages11
JournalHuman immunology
Issue number2
Publication statusPublished - 1 Feb 2003


  • Cellular peptide binding assay
  • Competition
  • Fluorescent peptide
  • HLA class I
  • MHC class I
  • Peptide binding

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